Protein Displacement Phenomena in Blood Plasma and Serum

Chapter 10

Downloaded by NORTH CAROLINA STATE UNIV on December 2, 2012 | http://pubs.acs.org Publication Date: May 5, 1995 | doi: 10.1021/bk-1995-0602.ch010

Protein Displacement Phenomena in Blood Plasma and Serum Studied by the Wettability Gradient Method and the Lens-on-Surface Method 1,3

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Hans B. Elwing , Liu Li , Agneta R. Askendal , Ghada S. Nimeri , and John L. Brash 2

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Interface Biology Group, Department of Physics and Measurement Technology, Linköping Institute of Technology, S-581 83 Linköping, Sweden Department of Chemical Engineering, McMaster University, Hamilton, Ontario L8S 4L7, Canada

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Protein exchange or displacement phenomena on silicon-based surfaces in heparinized blood plasma or serum was studied using the lens-on-surface method and the wettability gradient method. Ellipsometry was used for the quantification of adsorbed proteins. Selected proteins were detected with the use of antibodies. Using the lens-on-surface method it was found that the lack of FG (fibrinogen) in serum did not facilitate binding of anti-HSA (albumin), anti-IgG (immunoglobulin G) or anti-HMWK (high molecular weight kininogen) following incubation of serum with silicon oxide surfaces. Instead, the binding of these antibodies was virtually the same in serum as in plasma. Thus it seems that the behavior of fibrinogen in the "Vroman sequence" is essentially independent of these other proteins. With the wettability gradient method it was found that binding of anti-HSA increases with increasing incubation time at the hydrophobic end of the gradient when either plasma or serum was used. Anti-FG was found to bind to plasma-incubated gradient surfaces at both the hydrophobic and hydrophilic ends of the gradient at short incubation time. With prolonged incubation, binding of anti-FG decreased at the hydrophilic end of the gradient. Binding of anti-HMWK occurred mostly at the hydrophilic end of both plasma and serum incubated gradient surfaces with a binding optimum at about 10 min. With prolonged incubation the binding of anti-HMWK decreased. Binding of anti-IgG was relatively low on serum -incubated as well as plasma-incubated gradient surfaces. 3

Current address: Laboratory of Interface Biophysics, University of Göteborg, Lundberg Building, Medicinaregatan 9c, S-413 90 Göteborg, Sweden 0097-6156/95/0602-0138$12.00/0 © 1995 American Chemical Society In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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Solid surfaces are spontaneously covered with a layer of protein after seconds of contact with blood (1,2). Because this accumulation of protein occurs so rapidly, it precedes the arrival of cells on implant surfaces. Cells are therefore likely to interact with the protein coated biomaterial rather than directiy with the material. Therefore, much research has been done to understand the process of protein adsorption at solid surfaces. The physicochemical aspects of protein adsorption at solid surfaces have been investigated extensively with wide implications in different areas of research (3,4). Of particular interest in biomaterials oriented research is that many proteins change biological activity and conformation upon adsorption (5-9), and form an increasingly irreversible attachment to the surface with time (10,11). The subject of this investigation is the phenomenon of surface exchange or displacement of adsorbed proteins, an important effect in complex protein "solutions" like blood. This phenomenon has created much interest since Vroman's original observation of the "transitory" adsorption of fibrinogen from blood plasma on glass surface (12-18). According to Vroman, blood protein adsorption at glass surface has the characteristics of a sequence starting with albumin, followed by IgG, fibrinogen and finally high molecular weight kininogen (19). Additional proteins were included in the sequence in Vroman's original publication but these have been excluded in this investigation for reasons of simplicity. The "lens-on-surface" method was used by Vroman and was found to be very sensitive for these types of study (79). With this method we have been able to reproduce some of Vroman's original observations and to quantify objectively the displacement reaction with a supplementary ellipsometric method (20,21). The blood plasma concentration and the time of incubation have an influence on the speed of the displacement reaction (14,15,22). The degree of surface hydrophobicity is also an important factor since more hydrophobic surfaces bind proteins less reversibly (23,24). Therefore we have also investigated the "Vroman effect" in human plasma on the so-called wettability gradient surface (25), formed by diffusion of methylsilane over a silicon oxide surface. In this system we have demonstrated relations between both time of incubation and surface hydrophobicity and the exchange between fibrinogen and high molecular weight kininogen on gradient surfaces incubated in human plasma (26). Fibrinogen (FG) is an important protein in biomaterial interactions due to its many biological effects. It is therefore important to understand the adsorption of this protein in more detail. To this end we have studied the Vroman effect in fibrinogen-poor serum using the wettability gradient and lens-on-surface methods. By comparing the results of the serum and plasma experiments, it was hoped to get more information, both qualitative and quantitative, on the relative importance of albumin, IgG and H M W K in the "Vroman sequence". This work is based partly on original observations that H M W K could be detected at hydrophilic silicon surfaces incubated in serum. This observation was first reported by P. Warkentin at the San Diego meeting in 1984. See also Warkentin, et al., this volume. Materials and Methods Plasma, serum, and antibodies. Human heparinized plasma and serum were obtained from healthy donors and stored at -70°C until use. The plasma and serum were diluted to a desired concentration with phosphate buffered saline (PBS). Rabbit anti-human albumin (a-HSA), rabbit anti-human IgG (a-IgG), rabbit anti-human fibrinogen (a-FG), and rabbit anti-goat immunoglobulin were obtained from D A K O Immunoglobulin a/s, Glostrup, In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Denmark. Goat anti-human high molecular weight kininogen (a-HMWK) was from Nordic Immimochemical Laboratories, the Netherlands. The antibodies were diluted in PBS buffer to a working concentration of 1/50. The concentration of H M W K in plasma and serum was determined using a double diffusion immunoassay. No difference in precipitation ability between plasma and serum was found, indicating that H M W K in serum had at least 50% of its concentration in plasma. Preparation of wettability gradient surfaces. Polished silicon wafers, 0.3 mm thick (Okmetic O Y , Finland) were used as solid substrate. The wafers were cut into rectangles (10 x 25 mm) which were washed as previously described (25). Wettability gradient surfaces were prepared on silicon substrates arranged vertically in xylene (Merck p.a.). Dimethyldichlorosilane (0.05%, v/v, Sigma, USA) in 20 ml trichloroethylene (Merck p.a.) was added at the bottom and allowed to diffuse into the upper xylene phase for 90 minutes. The wettability gradient formed was approximately 7 mm long (25). The wettability properties can be determined by measuring the adsorption of fibrinogen (27), or by measuring the advancing water contact angle. The contact angle was found to vary from 90° at the hydrophobic end to 10° at hydrophilic end (Figure lb). Protein adsorption experiments on wettability gradient surfaces. The experiments were performed at room temperature (23°C). The surfaces were immersed in serum or plasma diluted 1/10 with PBS for 2.5 min, 10 min, 40 min, and 160 min, respectively. After rinsing with PBS, some of the surfaces were further incubated in the appropriate antibody solution for 30 minutes. The surfaces exposed to a-HMWK were then incubated in rabbit anti-goat immunoglobulin solution for 15 rnin in order to amplify the response of a-HMWK. Surfaces were finally rinsed in distilled water and dried in nitrogen. The lens-on-surface method. The details of this method have been described previously (20,28). Briefly, a glass lens with a focal length of about 200 mm was placed with the convex side down on the hydrophilic silicon surface. Serum or plasma (0.1 ml) at dilutions of 1/10 and 1/100 was injected into the space between the lens and the test surface. After 10 rnin incubation at room temperature, the plasma and the lens were removed. The test surface was then incubated in the appropriate antibody for 15 min after rinsing in PBS. The surfaces treated with a-HMWK were further incubated in rabbit anti-goat immunoglobulin for 15 min. All surfaces were finally rinsed with distilled water and dried in flowing nitrogen. Ellipsometric quantification of adsorbed proteins on surface. In ellipsometry the change in polarization of light reflected from a surface is measured, from which the thickness of the adsorbed film is calculated as described elsewhere (25). The dry gradient surfaces were monitored in an automatic ellipsometer (Auto Ell 3, Rudolph Research, N Y , USA) equipped with a device for stepwise lateral scanning. The resolution of the lateral measurements was 0.635 mm and scanning was performed stepwise at 0.635 mm intervals (Figure lc). The experiments presented in Figures 2-4 were done at least three times. No major qualitative differences between replicate experiments were found. Some properties of the silicon oxide and methylated silicon oxide surfaces. The silicon of electronic device quality used in this investigation has a layer of spontaneously grown, hydrophilic, silicon oxide. The top layer of the surface contains a large number of silanol groups which are amphoteric, being both proton acceptors and donors. The zeta

In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

In Proteins at Interfaces II; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995. 2

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Xylene Surface

Figure 1. Methods used in this investigation, a) The Lens-on-surface method. A convex glass lens is placed on a silicon oxide surface and blood plasma or serum is pipetted under the lens. After incubation the glass is removed from the surface. Detection of specific adsorbed proteins is made by subsequent incubation of the surface in antiserum, b) The wettability gradient surface is a silicon surface with a thin layer (5A) of silicon dioxide. Controlled hydrophobic gradients are made on these surfaces by diffusion of dichlorodimethylsilane. The gradient surfaces are incubated in plasma or serum. Detection of specific adsorbed proteins is made by subsequent incubation in antiserum, c) Quantification of protein adsorption and antibody adsorption is made by "scanning ellipsometry" across interesting sections of the surface.

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Figure 2. Adsorbed amount of organic material on silicon oxide surface as obtained by the lens on surface method. Plasma or serum diluted 1/10 or 1/100 was incubated under the lens for 10 min. After removal of the lens, the surfaces were incubated with specific antibodies for 15 min. Scanning ellipsometry determinations of the adsorbed amounts of organic material were performed across the contact point of the lens. The very high amounts of organic material deposited, about 0.8 M-g/cm , in the "a-HMWK" group may be explained by the fact that polyclonal anti-immunoglobulin was used to amplify the reaction. 2



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potential of these surfaces has been found to be zero at pH 3.0 to 3.2 (29). The silicon oxide surface is made hydrophobic by means of reaction with dicWorodimethylsilane (DDS). DDS reacts with the silanol groups of the surface and the resulting -Si(CH3)2 groups are bound covalently to the silicon oxide forming a densely packed array of methyl groups (30). Silicon of electronic device quality has a surface that is flat at the nanometer scale. The extreme flatness makes it highly reflective and thereby suitable as a substrate for ellipsometry. In a recent investigation of plasma protein adsorption on solid surfaces of different origin it was found that both silicon oxide and methylated silicon oxide generally adsorb less protein per unit area than some polymer surfaces, for example polystyrene (31). It is possible that the extreme flatness (as measured by scanning force microscopy) of the silicon surface is responsible for these differences (30). Results The lens-on surface method and the "Vroman effect" in serum and plasma. The principle of the lens method is that a gradient in fluid height or volume above the surface, is created from the contact point of the lens at the surface and outwards (Figure la). When serum or plasma is incubated under the lens, the protein solution close to the contact point will be depleted of protein due to adsorption at the surface. Such depletion will have an effect which is similar (but not identical) to dilution of the protein solution near the contact point of the lens (21). Serum or blood plasma diluted 1/10 and 1/100 were used in these experiments with the hydrophilic silicon surface. After washing off unbound protein and removal of the lens, the surfaces were incubated in various antisera followed by ellipsometric measurement of the amount of adsorbed protein. Representative data are shown in Figure 2. At a dilution of 1/10 binding of anti-HMWK occurred at distances far from the contact point of the lens. There was no apparent difference between plasma and serum in the binding pattern of anti-HMWK. Binding of anti-FG on surfaces incubated in plasma showed maxima at some distance from the contact point (2-3 mm) as previously observed (20). On surfaces incubated in serum, little binding of anti-FG was observed as expected. Binding of anti-HSA and anti-IgG was low on both plasma- and serum-incubated surfaces. In experiments with plasma and serum diluted 1/100 the binding of anti-HMWK was very similar. The total bound anti-HMWK was however lower than in the 1/10 dilution experiments. In addition, there were no binding maxima of anti-FG on plasmaincubated surfaces. The binding of anti-IgG or anti-HSA was insignificant in the 1/100 dilution experiments. The double peaks representing binding of anti-IgG on serumincubated surfaces were frequently, but not always, observed. Deposition of plasma and serum proteins on wettability gradient surfaces. The lens-on-surface method can be used only on hydrophilic surfaces; it is precluded on hydrophobic surfaces due to capillary effects. Investigation and assessment of protein exchange phenomena using the gradient method were done at different times of incubation. Gradient surfaces were incubated with plasma or serum, diluted 1/10, for 2.5 rnin, 10 rnin, 40 min and 160 min at room temperature. After rinsing and drying the surfaces, the adsorbed amounts of organic material were determined with the ellipsometer. The results of a representative experiment are shown in Figure 3. Approximately 0.25 pg/cm^ was deposited at the surface independent of the time of adsorption. However, at the


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Protein Displacement Phenomena in Blood Plasma and Serum

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